The differences between virulent and avirulent forms of a bacterial pathogen typically lie in a small set of genes that encode proteins called "virulence factors". The great success of recent genome and comparative genome sequencing efforts, together with powerful bioinformatics approaches, suggest that virulence factors will soon be identified for a broad range of human pathogens. Anti-toxin approaches that treat infection therapeutically by targeting key virulence factors should provide an excellent alternative or complement to antibiotics and vaccines. Recent experience with B. anthracis has demonstrated that antibiotics approaches to disease control are successful when implemented early in infection, but if diagnosed late the accumulated toxins retain their lethal effect; the need for anti-toxins in this case is thus clear-cut. Our current understanding of the molecular basis of anthrax intoxication, the fruits of many years of basic research, gives us many opportunities to design anti-toxins. In this project, we will build on our prior structural work on the anthrax lethal toxin in two principal directions. First, we will determine the structures of complexes between the anthrax toxin proteins and their host cell targets. Second, we will explore a new hypothesis on the structural basis of the CO2 trigger of anthrax virulence gene expression, which will involve determining crystal structures of key elements of the virulence plasmid transcriptional apparatus. Our bioinformatics approaches will continue to characterize new protein families to be tested. The Specific Aims are: (1) Crystal structures of anthrax Lethal Factor in complex with MEK1, and anthrax Protective Antigen in complex with its host cell receptor. (2) Structural studies of a putative CO2 sensor domain, "BACO", and its complexes with the "master regulator" of virulence transcription, AtxA. (3) Crystal structure of virulence factors in complex with inhibitors, to aid the scientific discovery of new virulence factors and inhibitor design. Together, these studies will increase our understanding of bacterial pathogenesis and provide valuable data for the design of drugs to treat NIAID priority pathogens.